CN107449900B - Multi-residue detection antigen and antibody of carbamate and pyrethroid, and preparation method and application thereof - Google Patents
Multi-residue detection antigen and antibody of carbamate and pyrethroid, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a carbamate and pyrethroid multi-residue detection antigen and antibody. The invention fills up the technical blank in the field for a long time, provides a combined antigen for simultaneously detecting multiple residues of carbamates and pyrethrins, and effectively establishes a detection method for simultaneously detecting multiple residues of carbamates and pyrethrins based on the combined antigen. Has wide application prospect. The carbamate and pyrethroid multi-residue detection antigen has a structural formula shown in a formula (I):
（Ⅰ）。
Description
Technical Field
The invention relates to the technical field of food safety, in particular to a multi-residue detection antigen and antibody of carbamate and pyrethroid, and a preparation method and application thereof.
Background
China is a big agricultural country, the yield and the consumption of agricultural products are in the forefront of the world every year, but the agricultural development of China is always puzzled by the problem of pesticide residues. Carbamate pesticides and pyrethroid pesticides are two types of pesticides common in agricultural production in China, so researchers develop a series of detection methods for the two types of pesticides, and immunoassay methods have the advantages of high sensitivity, strong specificity, simple pretreatment, short detection period, suitability for large-batch sample detection and the like, so that the immunoassay method becomes a hotspot of research in recent years. However, due to the limitation of specific thinking, the existing immunoassay method is only suitable for detecting one kind of pesticides, but cannot simultaneously detect a plurality of pesticides, thereby causing useless repeated labor and waste of resources. Therefore, the development of a method for simultaneously and rapidly detecting carbamate pesticides and pyrethroid pesticides is of great significance.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the technical blank of simultaneous detection and analysis of the residue of the existing carbamate pesticide and the pyrethroid pesticide and provides an antigen for simultaneously detecting the residue of the carbamate pesticide and the pyrethroid pesticide.
The invention also aims to solve the technical problem of a preparation method of the hapten and the antigen for simultaneously detecting the residue of the carbamate pesticide and the pyrethroid pesticide.
Another technical problem to be solved by the present invention is to provide an antibody for simultaneously detecting carbamate-based pesticide and pyrethroid-based pesticide residues.
The invention also aims to solve the technical problem of simultaneously detecting the antigens and the antibodies for the residue of the carbamate pesticide and the pyrethroid pesticide.
The purpose of the invention is realized by the following technical scheme:
provides a carbamate and pyrethroid combined antigen, the structural formula of which is shown as the formula (I):
preferably, the carrier protein is bovine serum albumin, human serum albumin, hemocyanin, or ovalbumin.
The invention also provides a preparation method of the carbamate and pyrethroid multi-residue antigen, which comprises the steps of coupling the carbamate hapten with carrier protein by adopting an active ester method, and then continuously coupling the coupled product with the pyrethroid hapten by the active ester method to prepare and obtain the carbamate and pyrethroid multi-residue detection antigen.
Specifically, the preparation method of the carbamate and pyrethroid combined antigen comprises the following steps:
s1, dissolving carbamate hapten in DMF to form a mixed solution, adding EDC and NHS, and stirring at room temperature for reaction; after the reaction is finished, adding the reaction solution into a phosphate (pH7.4) buffer solution of the carrier protein, continuing the reaction, and dialyzing the reaction solution at room temperature to prepare the complete carbamate antigen;
s2, dissolving the pyrethroid hapten in DMF to form a mixed solution, adding EDC and NHS, and stirring at room temperature for reaction;
s3, dissolving the complete carbamate antigen prepared in the step S1 in a DMF solution, adding the reaction solution prepared in the step S2, reacting at room temperature, and dialyzing the reaction solution after the reaction is finished to obtain the multi-residue detection antigen of carbamates and pyrethrins.
Preferably, the mass ratio of the carbamate hapten to the carrier protein in the step S1 is 12: 1.
Preferably, the reaction time of the carbamate hapten, EDC and NHS in the step S1 is 8-24 h.
Preferably, the reaction time of the carbamate hapten and the carrier protein in the step S1 is 6-24 h.
Preferably, the reaction time in the step S2 is 8-12 h.
Preferably, the mass ratio of the pyrethroid hapten to the carbamate antigen in the step S3 is 10: 1.
Preferably, the reaction time in the step S3 is 6-8 h.
In the above preparation method, the mass ratio of DMF, EDC and NHS was determined at 10:2: 1.
In the above preparation method, the composition of the phosphate buffer solution of carrier protein is 50mg BSA dissolved with 6mL PBS, and then 3.75mL LDMF is added, the dosage is 50 mg.
Preferably, the carrier protein is bovine serum albumin, human serum albumin, hemocyanin, or ovalbumin.
The carbamate hapten has a molecular structure shown in a formula (II):
the pyrethroid hapten has a molecular structure shown in a formula (III):
preferably, the carbamate hapten is prepared by the following steps:
s11, dissolving furan phenol in dichloromethane, adding pyridine to form a mixed solution, then dropwise adding a p-nitrophenyl chloroformate solution (the p-nitrophenyl chloroformate is dissolved in the dichloromethane) into the mixed solution, keeping the whole system at a temperature of below 4 ℃ in a salt bath, after the reaction is finished, oscillating and washing the reaction solution with dilute hydrochloric acid, and purifying the reaction solution by a column to obtain a product; the product is a light yellow crystal;
s12, dissolving the product obtained in the step S11 in tetrahydrofuran to form a tetrahydrofuran mixed solution, dissolving 4-aminobutyric acid in a sodium bicarbonate solution, dropwise adding the tetrahydrofuran mixed solution into the sodium bicarbonate solution in ice bath, after the reaction is finished, continuously adding dilute hydrochloric acid in the ice bath to adjust the pH of the reaction solution to 4.0, extracting the reaction solution with ethyl acetate, adding anhydrous sodium sulfate into the extracting solution for dehydration, concentrating, performing conventional column chromatography purification, collecting a target component, and removing the solvent through reduced pressure distillation to obtain the carbamate hapten.
Preferably, the number of times of extracting the reaction solution with ethyl acetate in step S12 is 3.
Preferably, the pyrethroid hapten is prepared by the following method:
s21, mixing carbon tetrachloride, acetonitrile and water according to the weight ratio of 10: mixing according to the volume ratio of 10:15 to form a mixture of carbon tetrachloride, acetonitrile and water for later use;
s22, adding 2.2g of cypermethrin, 0.12g of ruthenium trichloride, 4.3g of sodium periodate and 35mL of the prepared mixed solution into a reaction bottle, heating, reacting, evaporating an organic solvent after the reaction is finished, adjusting the pH value of the residual solution to be acidic, extracting with ethyl acetate, and washing with water; purifying the organic phase by a column to obtain the pyrethroid hapten.
Preferably, the heating temperature in step S22 is 60 ℃, and the reaction time is 2 h.
The invention also provides application of the carbamate and pyrethroid multi-residue antigen in preparing carbamate and pyrethroid multi-residue antibodies or detecting carbamate and pyrethroid pesticide residues.
The invention also provides a carbamate and pyrethroid multi-residue detection antibody prepared by adopting the carbamate and pyrethroid multi-residue detection antigen, and application of the antibody in detecting carbamates and pyrethrin.
The invention has the beneficial effects that:
the invention fills up the technical blank in the field for a long time, provides a combined antigen for simultaneously detecting the multiple residues of the carbamates and the pyrethrins, effectively establishes a detection method for simultaneously detecting the multiple residues of the carbamates and the pyrethrins based on the combined antigen, and has wide application prospect.
In order to realize the purpose of the invention, the invention simultaneously provides two haptens and a preparation method of the haptens, and the specific antigens with multi-residue carbamates and pyrethrins can be prepared very simply and easily based on the two haptens, thereby laying a technical foundation for the detection technical scheme of the invention.
The preparation method has mild conditions and is easy to popularize.
Drawings
FIG. 1 scheme for synthesis of carbamate haptens.
FIG. 2 shows the mass spectrum of carbamate hapten.
FIG. 3 scheme for synthesis of pyrethroids haptens.
FIG. 4 mass spectrum of pyrethroid hapten.
FIG. 5A standard curve for the carboweilicELISA.
FIG. 6 Cypermethrin ICELISA Standard Curve.
Detailed Description
The process of the present invention is further illustrated below with reference to specific examples. The following examples are for illustrative purposes only and are not to be construed as limiting the invention. Unless otherwise specified, the biomaterials, reagent raw materials used in the following examples are conventionally commercially available or commercially available biomaterials and reagent raw materials, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.
EXAMPLE 1 preparation of Carbamate and pyrethroid haptens
(1) Synthesis of carbamate haptens
S11, weighing 0.8g of furan phenol, dissolving the furan phenol in 15mL of dichloromethane, adding 0.58mL of pyridine to form a mixed solution, dropwise adding 1g of p-nitrophenylchloroformate solution (dissolved in 10mL of dichloromethane) into the mixed solution, and reacting for 3 hours at 4 ℃ in an ice salt bath; after the reaction is finished, the reaction solution is shaken and washed in a separating funnel by using 3% hydrochloric acid, and the washing solution is purified by passing through a column to obtain a light yellow crystal.
S12, taking 0.4g of the reaction product, dissolving the reaction product in 12mL of tetrahydrofuran, taking 0.16g of 4-aminobutyric acid, dissolving the 4-aminobutyric acid in 12mL of sodium bicarbonate solution with pH8.2, then dropwise adding the tetrahydrofuran solution into the sodium bicarbonate solution under ice bath, and reacting for 4.5h at room temperature. After the reaction is finished, continuously adding 4mol/L hydrochloric acid solution under ice bath, adjusting the pH of the reaction solution to 4, extracting the reaction solution for 3 times by using ethyl acetate, dehydrating an organic phase by using anhydrous Na2SO4, and then, purifying by using a column, and distilling under reduced pressure to remove the solvent to obtain the amino acid methyl ester hapten. The mass spectrum of the amino acid methyl ester hapten is shown in figure 2. The molecular structure shown in the formula (II) is proved to be:
(2) synthesis of pyrethrin hapten
S21, mixing carbon tetrachloride, acetonitrile and water according to the weight ratio of 10: 10:15 to form a mixed solution of carbon tetrachloride, acetonitrile and water;
s22, taking 2.2g of cypermethrin, 0.12g of ruthenium trichloride, 4.3g of sodium periodate and 35mL of the mixed solution, heating to 60 ℃ for reaction for 2 hours, evaporating the organic solvent after the reaction is finished, adjusting the pH value of the residual solution to acidity, extracting with ethyl acetate, washing with water, taking the organic phase, and purifying by a column to obtain the pyrethroid hapten. The mass spectrum of the pyrethroid hapten is shown in figure 4. The molecular structure shown in the formula (III) is proved to be:
EXAMPLE 2 preparation of Carbamate and pyrethroid combination antigens
S1, weighing 29mg of the carbamate hapten prepared in example 1, dissolving the carbamate hapten in 1mL of DMF, stirring, adding 36mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 25mg of N-N-hydroxysuccinimide (NHS), stirring at room temperature for reacting for 8 hours, after the reaction is finished, adding 0.25mL of reaction liquid into 50mg of pH7.4BSA phosphate buffer solution, stirring at room temperature for 6 hours, then filling the reaction liquid into a dialysis bag, dialyzing for 3 days by using the pH7.4 phosphate buffer solution, and changing water for 2-3 times every day to prepare a carbamate complete antigen;
s2, weighing 37mg of the pyrethroid hapten prepared in the embodiment 1, dissolving the pyrethroid hapten in 1mL of DMF, stirring, adding 36mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 25mg of N-N-hydroxysuccinimide (NHS), and stirring at room temperature for reacting for 8 hours to obtain a reaction solution;
s3, adding 0.25mL of reaction solution into a DMF (dimethyl formamide) solution of complete carbamate antigens, stirring at room temperature for 6 hours, then filling into a dialysis bag, dialyzing for 3 days by using a phosphate buffer solution with the pH value of 7.4, changing water for 2-3 times every day, and obtaining the multi-residue detection antigens of carbamates and chrysanthemums after dialysis is finished.
EXAMPLE 3 preparation and characterization of antibodies
Mixing the immunogen with an equal dose of immune adjuvant (Freund complete adjuvant for 1 st immunization and Freund incomplete adjuvant for military boosting immunization later), completely emulsifying by using a stirrer, carrying out multipoint immunization on the back of a healthy white rabbit, boosting immunization once every two weeks later, measuring the serum titer and the inhibition rate by using indirect ELISA, and boosting immunization once again after the titer is stable. Blood is collected from the heart 10 days after the last immunization, and serum is retained by centrifugation. And purifying the serum by adopting an octanoic acid-ammonium sulfate salting-out method to obtain the antibody with high specificity.
Example 4 establishment of enzyme-linked immunosorbent assay
The working concentration of antigen and antibody was determined by matrix titration with the working concentration of coating antigen being 0.5. mu.g/mL and the dilution factor of carbamate and pyrethroid antibody being 1/40000.
The experimental solutions were prepared from carbamate and pyrethroid solutions at different concentrations, and competition was performed under gradient dilution using 9 parallel experiments (n-3). Indirect competitive ELISA method: with the coated ELIAS plate of above-mentioned working concentration, add experiment solution and antibody solution simultaneously to the ELIAS plate aperture, set up blank hole and negative control hole simultaneously, 37 ℃ incubation 40min, pour out downthehole liquid, wash 5 times with the washing liquid, invert the ELIAS plate and pat on absorbent paper: adding diluted enzyme-labeled secondary antibody solution, incubating for 20min at 37 ℃, washing for 5 times by using washing liquid, patting dry: adding substrate color developing solution, incubating at 37 deg.C for 10min, adding stop solution to stop color development, measuring absorbance OD at wavelength of 450nm with microplate reader, taking absorbance OD as ordinate, taking log10 value of carbamate and pyrethrin experimental solution concentration as abscissa, drawing semilogarithmic standard curve, and referring to competition standard curve of carbamate and pyrethrin ELISA (taking carbofuran and cypermethrin as examples respectively) shown in figure 5 or figure 6.
The result shows that the standard curve has a complete reverse S shape and is provided with an upper platform and a lower platform, the parallel determination times of the standard curve are 3 times, the experimental repeatability is good, and the relative standard deviation is within 15 percent.
A standard curve is drawn by the same method to calculate the lowest limit of detection (LOD value) and half-maximal Inhibition (IC) of other carbamate and pyrethroid pesticides50Values), linear range, as shown in table 1. The method has better accuracy and sensitivity, and can be used for detecting carbamate and pyrethrin in agricultural products and food samples.
TABLE 1 LOD, IC for 5 drugs detected by antibody50LR value
| Name of drug | LOD(mg/L) | IC50(mg/L) | LR(mg/L) |
| Carbofuran | 0.51 | 61.1 | 6.6～521.8 |
| Cypermethrin | 1.24 | 74.77 | 7.88～519.50 |
| Deltamethrin | 3.3 | 143.2 | 13.2～1556.0 |
| Fenpropathrin | 7.54 | 531.9 | 15.50～1824.8 |
| Biphenthrin | 14.21 | 824.3 | 27.19～2499.1 |
Claims (10)
1. A multi-residue detection antigen of carbamate and pyrethroid has a structural formula shown in formula (I):
the carrier protein is bovine serum albumin.
2. The method for preparing the multi-residue detection antigen of carbamates and pyrethrins as claimed in claim 1, wherein the preparation method comprises the steps of coupling the carbamate hapten with bovine serum albumin by an active ester method, and then continuously coupling the coupled product with the pyrethroid hapten by the active ester method to prepare the multi-residue detection antigen of carbamates and pyrethrins.
3. The method according to claim 2, characterized in that it comprises the following steps:
s1, dissolving carbamate hapten in DMF to form a mixed solution, adding EDC and NHS, and stirring at room temperature for reaction; after the reaction is finished, adding the reaction solution into a phosphate buffer solution of carrier protein with pH of 7.4, continuing the reaction, and dialyzing the reaction solution at room temperature to prepare a complete carbamate antigen;
s2, dissolving the pyrethroid hapten in DMF to form a mixed solution, adding EDC and NHS, and stirring at room temperature for reaction;
s3, dissolving the complete carbamate antigen prepared in the step S1 in a DMF solution, adding the reaction solution prepared in the step S2, reacting at room temperature, and dialyzing the reaction solution after the reaction is finished to obtain the multi-residue detection antigen of carbamates and pyrethrins;
the carbamate hapten has a molecular structure shown in a formula (II):
the pyrethroid hapten has a molecular structure shown in a formula (III):
4. the method according to claim 3, wherein the mass ratio of the carbamate hapten to the carrier protein in the step S1 is 12: 1; the reaction time of the carbamate hapten, EDC and NHS in the step S1 is 8-24 h; and S1, the reaction time of the carbamate hapten and the carrier protein is 6-24 h.
5. The method according to claim 3, wherein the reaction time of step S2 is 8-12 h; the mass ratio of the pyrethroid hapten to the carbamate complete antigen in the step S3 is 10: 1; step S3, the reaction time is 6-8 h; the mass ratio of DMF, EDC and NHS was determined as 10:2: 1.
6. The method of claim 3, wherein the carbamate hapten is prepared by:
s11, dissolving furan phenol in dichloromethane, adding pyridine to form a mixed solution, dropwise adding a p-nitrophenylchloroformate solution dissolved in dichloromethane into the mixed solution, keeping the whole system at a temperature below 4 ℃ in a salt bath, after the reaction is finished, oscillating and washing the reaction solution with dilute hydrochloric acid, and purifying the reaction solution by passing through a column to obtain a product;
s12, dissolving the product obtained in the step S11 in tetrahydrofuran to form a tetrahydrofuran mixed solution, dissolving 4-aminobutyric acid in a sodium bicarbonate solution, dropwise adding the tetrahydrofuran mixed solution into the sodium bicarbonate solution in ice bath, after the reaction is finished, continuously adding dilute hydrochloric acid in the ice bath to adjust the pH of the reaction solution to 4.0, extracting the reaction solution with ethyl acetate, adding anhydrous sodium sulfate into the extracting solution for dehydration, concentrating, performing column purification, collecting a target component, and removing the solvent through reduced pressure distillation to obtain the carbamate hapten.
7. The method of claim 3, wherein the pyrethroid hapten is prepared by:
s21, mixing carbon tetrachloride, acetonitrile and water according to the weight ratio of 10: mixing according to the volume ratio of 10:15 to form a mixture of carbon tetrachloride, acetonitrile and water for later use;
s22, adding 2.2g of cypermethrin, 0.12g of ruthenium trichloride, 4.3g of sodium periodate and 35mL of the prepared mixed solution into a reaction bottle, heating, reacting, evaporating an organic solvent after the reaction is finished, adjusting the pH value of the residual solution to be acidic, extracting with ethyl acetate, and washing with water; purifying the organic phase by a column to obtain the pyrethroid hapten.
8. Use of the carbamate and pyrethroid multi-residue antigen of claim 1 or the carbamate and pyrethroid multi-residue antigen prepared by the method of claims 2 to 7 in the preparation of carbamate and pyrethroid multi-residue antibodies or in the detection of carbamate and pyrethroid pesticide residues.
9. Carbamate and pyrethroid multi-residual antibodies prepared by using carbamate and pyrethroid multi-residual antigens according to claim 1 or carbamate and pyrethroid multi-residual antigens prepared by the method according to claims 2 to 7.
10. Use of the multi-residue antibodies of carbamates and pyrethrins according to claim 9 for the detection of carbamates and pyrethrins.
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| CN115611774B (en) * | 2022-09-26 | 2024-04-12 | 华南农业大学 | Fenpropathrin hapten, artificial antigen and fenpropathrin detection method |
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